To elucidate the mechanism for a reported cAMP-dependent stimulation of calcium uptake by smooth muscle microsomes, fast kinetic analyses of reaction steps involved in calcium transport by endoplasmic reticulum vesicles (ER) from bovine aorta muscularis will be undertaken. Initially, studies will be devoted to the identification of ER proteins phosphorylated by cAMP-dependent protein kinase and the effect of this phosphorylation on the calcium dependence of oxalate-supported calcium uptake. It is postulated that cAMP-dependent phosphorylation of a 44K dalton protein will increase the affinity of the transport mechanism for calcium; hence, transient state kinetic studies, which require substantial amounts of protein, will be done at Km for calcium of control ER. Parameters to be examined in these transient kinetic studies will include rate of phosphointermediate (EP) formation, EP decay and calcium binding to EP, where EP is distinguishable from the cAMP-dependent phosphorylated protein(s) on the basis of hydroxylamine sensitivity, calcium dependence, and molecular weight. The time course for these transient state kinetic studies is on the order of msec; therefore, a Commonwealth Technology quench flow apparatus (capable of 4-400 msec resolution) will be employed. It is expected that cAMP-dependent phosphorylation will greatly increase the rate of EP decay, with less stimulation of EP formation rate and calcium binding rate, as in the case of cardiac sarcoplasmic reticulum (SR). Thus, the above experiments will define the mechanism by which cAMP-dependent phosphorylation alters calcium transport by vascular ER as well as allowing a comparison with the cardia SR calcium transport system. Furthermore, extending these studies to include cGMP and calmodulin-dependent proten kinase effects may permit elucidation of the role of ER in maintenance of vasclar tone. Clearly, however, future studies must also include ER from resistance vessels (e.g., mesenteric arterioles).